Immune Cell Biomarkers of Cardiovascular Disease

Abstract

The present invention includes methods to determine a risk of a cardiovascular disease (CVD) or a subclinical CVD (sCVD) in a subject, by detecting a subset of intermediate monocyte cell populations in the immune cells, wherein a percentage of the subsets as compared to a total intermediate monocyte cell population present in the isolated immune cells is determined; comparing a proportion of the subsets subset of intermediate monocyte cell populations determined with a statistical sample representative of a proportion of equivalent subsets in a total intermediate monocyte cell populations from a subject that does not have a cardiovascular disease; and determining that the subject has an increased risk for cardiovascular disease where the subject has a decrease in the certain subsets, has an increase in another subset, or both, as compared to the statistical sample.

What is claimed is: 
     
         1 . A method to determine a risk of a cardiovascular disease (CVD) or a subclinical CVD (sCVD) in a subject, the method comprising:
 (a) obtaining a sample from the subject;   (b) isolating immune cells from the sample;   (c) detecting a first (INT1), second (INT2), third (INT3) and fourth (INT4) subset of intermediate monocyte cell populations in the immune cells, wherein a percentage of the INT1, INT2, INT3, and INT4 subsets as compared to a total intermediate monocyte cell population present in the isolated immune cells is determined;   (d) comparing a proportion of the INT1, INT2, INT3, and INT4 subsets subset of intermediate monocyte cell populations determined in (c) with a statistical sample representative of a proportion of equivalent INT1, INT2, INT3, and INT4 subsets in a total intermediate monocyte cell populations from a subject that does not have a cardiovascular disease; and   (e) determining that the subject has an increased risk for cardiovascular disease where the subject has a decrease in the INT2 and INT3 subsets, has an increase in the INT4 subset, or both, as compared to the statistical sample.   
     
     
         2 . The method of  claim 1 , wherein if the subject is at increased risk based on the comparing step, then further treating the subject for cardiovascular disease. 
     
     
         3 . The method of  claim 1 , wherein the intermediate monocyte cell population is detected in the isolated immune cells by gating for CD3−CD19−CD14+CD16+CD56− immune cells. 
     
     
         4 . The method of  claim 1 , wherein the INT2 subset is detected by gating the isolated immune cells for IL6RA+, CD69+, CD86+, CD9+, CD141+, CD142+, CD163+, CCR2+, CCR4+, CCR5+, CD36+, CD38+, CD45RO+, CD206+. 
     
     
         5 . The method of  claim 1 , wherein the INT3 subset is detected by gating the isolated immune cells for IL6RA, CD141+, CD142+, CD163+, CCR2+, CD36+, CD38+, CD69+, CD86+CD9+, CD154, CCR4+, CD69+, and optionally LAG3+, CD206+, CD137+, CCR4+, CCR5+, CD206, CD45RO+, CD4. 
     
     
         6 . The method of  claim 1 , wherein the INT4 subset is detected by gating the isolated immune cells for IL6RA low , CD137 lower than INT 2 and 3 , CD154−, CD163 low , CCR2 low , CCR4 low , CCR5 low , CD206 low , LAG3−, CD36 lower than INT2 and 3 , CD38 low , and optionally CD45RA, CD9+. 
     
     
         7 . The method of  claim 1 , wherein the subject is determined to have a decrease in the INT2 subset as compared to the statistical sample, where the proportion of the INT2 subset is 40% of a total CD3−CD19−CD14+CD16+CD56− INT population in the statistical sample and less than 20% of a total CD3−CD19−CD14+CD16+CD56− INT population in the subject. 
     
     
         8 . The method of  claim 1 , wherein the subject is determined to have a decrease in the INT2 subset as compared to the statistical sample where there is one half of an amount of INT2 cells present in the subject as compared to the statistical sample. 
     
     
         9 . The method of  claim 1 , wherein the subject is determined to have a decrease in the INT3 subset as compared to the statistical sample, where the proportion of the INT3 subset is 30% of a total CD3−CD19−CD14+CD16+CD56− INT population in the statistical sample and 10% of the total CD3−CD19−CD14+CD16+CD56− INT population in the subject. 
     
     
         10 . The method of  claim 1 , wherein the subject is determined to have a decrease in the INT3 subset as compared to the statistical sample where there is one-third of an amount of INT3 cells present in the subject as compared to the statistical sample. 
     
     
         11 . The method of  claim 1 , wherein the subject is determined to have an increase in the INT4 subset as compared to the statistical sample, where the proportion of the INT4 subset is 12% of a total CD3−CD19−CD14+CD16+CD56− INT population in the statistical sample and 40% of a total CD3−CD19−CD14+CD16+CD56− INT population in the subject. 
     
     
         12 . The method of  claim 1 , wherein the subject is determined to have an increase in the INT4 subset as compared to the statistical sample where there is roughly four times an amount INT4 cells present in the subject as compared to the statistical sample. 
     
     
         13 . The method of  claim 1 , wherein the sample is a blood, serum, or plasma sample, and biomarkers are detected as a protein or a nucleic acid. 
     
     
         14 . A method for treating a cardiovascular disease (CVD) or a subclinical CVD (sCVD) in a subject, the method comprising:
 (a) obtaining a sample from the subject;   (b) isolating immune cells from the sample;   (c) detecting a first (INT1), second (INT2), third (INT3) and fourth (INT4) subset of intermediate monocyte cell populations in the immune cells, wherein a percentage of the INT1, INT2, INT3, and INT4 subsets as compared to a total intermediate monocyte cell population present in the isolated immune cells is determined;   (d) comparing a proportion of the INT1, INT2, INT3, and INT4 subsets subset of intermediate monocyte cell populations determined in (c) with a statistical sample representative of the proportion of equivalent INT1, INT2, INT3, and INT4 subsets in the total intermediate monocyte cell populations from a subject that does not have a cardiovascular disease;   (e) determining that the subject has a CVD or subclinical CVD, wherein if the subject has a decrease in the INT2 and INT3 subsets, has an increase in the INT4 subset, or both, as compared to the statistical sample, it is determined that the subject has a CVD or a subclinical CVD; and   (g) where the subject has a CVD or a subclinical CVD based on the comparing step, then further providing the subject with a treatment that prevents or treats the cardiovascular disease.   
     
     
         15 . The method of  claim 14 , wherein the intermediate monocyte cell population is detected in the isolated immune cells by gating for CD3−CD19−CD14+CD16+CD56− immune cells. 
     
     
         16 . The method of  claim 14 , wherein the INT2 subset is detected by gating the isolated immune cells for IL6RA+, CD69+, CD86+, CD9+, CD141+, CD142+, CD163+, CCR2+, CCR4+, CCR5+, CD36+, CD38+, CD45RO+, CD206+. 
     
     
         17 . The method of  claim 14 , wherein the INT3 subset is detected by gating the isolated immune cells for IL6RA, CD141+, CD142+, CD163+, CCR2+, CD36+, CD38+, CD69+, CD86+CD9+, CD154, CCR4+, CD69+, and optionally LAG3+, CD206+, CD137+, CCR4+, CCR5+, CD206, CD45RO+, CD4. 
     
     
         18 . The method of  claim 14 , wherein the INT4 subset is detected by gating the isolated immune cells for IL6RA low , CD137 lower than INT 2 and 3 , CD154−, CD163 low , CCR2 low , CCR4 low , CCR5 low , CD206 low , LAG3−, CD36 lower than INT2 and 3 , CD38 low , and optionally CD45RA, CD9+. 
     
     
         19 . The method of  claim 14 , wherein the subject is determined to have a decrease in the INT2 subset as compared to the statistical sample, where the proportion of the INT2 subset is 40% of a total CD3−CD19−CD14+CD16+CD56− INT population in the statistical sample and less than 20% of a total CD3−CD19−CD14+CD16+CD56− INT population in the subject. 
     
     
         20 . The method of  claim 14 , wherein the subject is determined to have a decrease in the INT2 subset as compared to the statistical sample where there is one half of an amount of INT2 cells present in the subject as compared to the statistical sample. 
     
     
         21 . The method of  claim 14 , wherein the subject is determined to have a decrease in the INT3 subset as compared to the statistical sample, where the proportion of the INT3 subset is 30% of a total CD3−CD19−CD14+CD16+CD56− INT population in the statistical sample and 10% of the total CD3−CD19−CD14+CD16+CD56− INT population in the subject. 
     
     
         22 . The method of  claim 14 , wherein the subject is determined to have a decrease in the INT3 subset as compared to the statistical sample where there is one-third of an amount of INT3 cells present in the subject as compared to the statistical sample. 
     
     
         23 . The method of  claim 14 , wherein the subject is determined to have an increase in the INT4 subset as compared to the statistical sample, where the proportion of the INT4 subset is 12% of a total CD3−CD19−CD14+CD16+CD56− INT population in the statistical sample and 40% of a total CD3−CD19−CD14+CD16+CD56− INT population in the subject. 
     
     
         24 . The method of  claim 14 , wherein the subject is determined to have an increase in the INT4 subset as compared to the statistical sample where there is roughly four times an amount INT4 cells present in the subject as compared to the statistical sample. 
     
     
         25 . The method of  claim 14 , wherein the sample is a blood, serum, or plasma sample, and biomarkers are detected as a protein or a nucleic acid. 
     
     
         26 . A method of diagnosing a cardiovascular disease (CVD) or a subclinical CVD (sCVD) in a subject, the method comprising:
 (a) obtaining a sample from the subject;   (b) isolating immune cells from the sample;   (c) detecting a first (INT1), second (INT2), third (INT3) and fourth (INT4) subset of intermediate monocyte cell populations in the immune cells, wherein a percentage of the INT1, INT2, INT3, and INT4 subsets as compared to a total intermediate monocyte cell population present in the isolated immune cells is determined;   (d) comparing a proportion of the INT1, INT2, INT3, and INT4 subsets subset of intermediate monocyte cell populations determined in (c) with a statistical sample representative of the proportion of equivalent INT1, INT2, INT3, and INT4 subsets in the total intermediate monocyte cell populations from a subject that does not have a cardiovascular disease; and   (e) determining that the subject has a cardiovascular disease or subclinical CVD, wherein if the subject has a decrease in the INT2 and INT3 subsets, has an increase in the INT4 subset, or both, as compared to the statistical sample, it is determined that the subject will develop or has a cardiovascular disease.   
     
     
         27 . The method of  claim 26 , wherein the intermediate monocyte cell population is detected in the isolated immune cells by gating for CD3−CD19−CD14+CD16+CD56− immune cells. 
     
     
         28 . The method of  claim 26 , wherein the INT2 subset is detected by gating the isolated immune cells for IL6RA+, CD69+, CD86+, CD9+, CD141+, CD142+, CD163+, CCR2+, CCR4+, CCR5+, CD36+, CD38+, CD45RO+, CD206+. 
     
     
         29 . The method of  claim 26 , wherein the INT3 subset is detected by gating the isolated immune cells for IL6RA, CD141+, CD142+, CD163+, CCR2+, CD36+, CD38+, CD69+, CD86+CD9+, CD154, CCR4+, CD69+, and optionally LAG3+, CD206+, CD137+, CCR4+, CCR5+, CD206, CD45RO+, CD4. 
     
     
         30 . The method of  claim 1 , wherein the INT4 subset is detected by gating the isolated immune cells for IL6RA low , CD137 lower than INT 2 and 3 , CD154−, CD163 low , CCR2 low , CCR4 low , CCR5 low , CD206 low , LAG3−, CD36 lower than INT2 and 3 , CD38 low , and optionally CD45RA, CD9+. 
     
     
         31 . The method of  claim 26 , wherein the subject is determined to have a decrease in the INT2 subset as compared to the statistical sample, where the proportion of the INT2 subset is 40% of a total CD3−CD19− CD14+CD16+CD56− INT population in the statistical sample and less than 20% of a total CD3−CD19−CD14+CD16+CD56− INT population in the subject. 
     
     
         32 . The method of  claim 26 , wherein the subject is determined to have a decrease in the INT2 subset as compared to the statistical sample where there is one half of an amount of INT2 cells present in the subject as compared to the statistical sample. 
     
     
         33 . The method of  claim 26 , wherein the subject is determined to have a decrease in the INT3 subset as compared to the statistical sample, where the proportion of the INT3 subset is 30% of a total CD3−CD19−CD14+CD16+CD56− INT population in the statistical sample and 10% of the total CD3−CD19−CD14+CD16+CD56− INT population in the subject. 
     
     
         34 . The method of  claim 26 , wherein the subject is determined to have a decrease in the INT3 subset as compared to the statistical sample where there is one-third of an amount of INT3 cells present in the subject as compared to the statistical sample. 
     
     
         35 . The method of  claim 26 , wherein the subject is determined to have an increase in the INT4 subset as compared to the statistical sample, where the proportion of the INT4 subset is 12% of a total CD3−CD19−CD14+CD16+CD56− INT population in the statistical sample and 40% of a total CD3−CD19−CD14+CD16+CD56− INT population in the subject. 
     
     
         36 . The method of  claim 26 , wherein the subject is determined to have an increase in the INT4 subset as compared to the statistical sample where there is roughly four times an amount INT4 cells present in the subject as compared to the statistical sample. 
     
     
         37 . The method of  claim 26 , wherein the sample is a blood, serum, or plasma sample, and biomarkers are detected as a protein or a nucleic acid. 
     
     
         38 . A method to determine whether a subject needs treatment for a cardiovascular disease (CVD) or a subclinical CVD (sCVD), the method comprising:
 (a) obtaining a sample from the subject;   (b) isolating the immune cells from the sample;   (c) measuring the presence of one or more immune cell subpopulations from the isolated immune cells, and an expression level of one or more biomarkers in the one or more immune cell subpopulations;   (d) comparing the expression level of the one or more biomarkers in (c) with a statistical sample representative of the biomarkers present in an equivalent immune cell subpopulation of a subject not having a cardiovascular disease; and   (e) determining that the subject is to be treated for the cardiovascular disease based on the comparing step of (d), wherein if the expression level of the one or more biomarkers present in the subject sample is higher than a baseline level of the statistical sample, the subject is determined to need treatment for the CVD or subclinical CVD.   
     
     
         39 . The method of  claim 38 , further comprising treating the subject in need of treatment for cardiovascular disease or subclinical CVD. 
     
     
         40 . The method of  claim 38 , wherein the one or more immune cell subpopulations is selected from CD4 T cells or subpopulations thereof, CD8 T cells or subpopulations thereof, intermediate monocytes or subpopulations thereof, B cells or subpopulations thereof, and Natural Killer cells or subpopulations thereof. 
     
     
         41 . The method of  claim 38 , wherein the sample is a blood, serum, or plasma sample, and the biomarkers are detected as a protein or a nucleic acid. 
     
     
         42 . The method of  claim 38 , wherein the immune cell subpopulation consists essentially of T cells and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 biomarkers selected from: SELPLG; IFITM3; MYC; FYB; JUNB; IL32; SELL; CCR7; GIMAP5; TCF7; FAS; BCL6; TNFSF10; STAT3; CD27; BIRC3; IKZF1; PTPRC; CYTIP; IFITM2; CD2; LCK; IL7R; FOXO1; TRAC; CCA; CD69; ICOS; SLC2A3; LEF1; RGS1; IL4R; CD52; HOPX; GZMA; KLRG1; KLRD1; TIGIT; STAT1; KLRC1; LGALS1; KLRC4; CD63; SAMD3; S100A10; KLRK1; IL23R; CCL5; APOBEC3G; CD160; TARP; CD3D; CD8A; KLRD1; ITGA4; CX3CR1; VNN2; GZMA; CH3L2; or GIMAP5, and optionally the biomarkers are selected in the order listed. 
     
     
         43 . The method of  claim 38 , wherein the immune cell subpopulation consists essentially of classical monocytes and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 40 biomarkers selected from: CCL4; CCL3; SLC2A3; SOD2; SELPLG; CD14; LAP3; FYB; LYN; CD80; TNFSF10; TNFSF8; CSF3; CSF3R; CD36; NAIP; SELL; CLEC4E; IL8R; PTPRC; VCAN; ADGRE1; MNDA; DUSP1; FYB; DUSP2; LYZ; JUNB; SCREP1; DOCK8; CD300A; LAP3; TNFSF13; SDCP; MDX1; FYN; STAT6; IL1B; NAMPT; STAT3; IL6; IER3; TLR2; CD83; FYB; S100A9; TNFSF13B; CLEC4E; or ICAM1, and optionally the biomarkers are selected in the order listed. 
     
     
         44 . The method of  claim 38 , wherein the immune cell subpopulation consists essentially of intermediate monocytes and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 biomarkers selected from: PRDM1; TNF; CCL4; LYZ; DUSP1; IL1B; DUSP2; CCL3; IER3; or ICAM1, and optionally the biomarkers are selected in the order listed. 
     
     
         45 . The method of  claim 38 , wherein the immune cell subpopulation consists essentially of B cells and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9 biomarkers selected from: CD74; IGHD; CD79A; LYN; CD83; R3HDM4; CD79B; CXCR5; or SLC2A3, and optionally the biomarkers are selected in the order listed. 
     
     
         46 . The method of  claim 38 , wherein the cardiovascular disease is selected from at least one of: coronary heart disease (CHD), heart failure (HF), coronary heart disease (CHD), cerebrovascular disease, peripheral arterial disease, atherosclerosis, or heart failure. 
     
     
         47 . A method to prevent cardiovascular disease (CVD) in a subject with unknown CVD status, the method comprising:
 (a) obtaining a sample from the subject;   (b) isolating immune cells from the sample;   (c) measuring a presence of one or more immune cell subpopulations from the isolated immune cells, and determining an expression level of one or more biomarkers in the one or more immune cell subpopulations;   (d) comparing the expression level of the one or more biomarkers in (c) with a statistical sample representative of the biomarkers in an equivalent immune cell subpopulation of a subject not having a cardiovascular disease;   (e) determining the CVD status of the subject based on the comparing step of (d), wherein if the expression level of the one or more biomarkers present in the subject sample is higher than a baseline level of the statistical sample, the subject is determined to need treatment to prevent CVD; and   (f) administering a treatment to prevent CVD in the subject.   
     
     
         48 . The method of  claim 47 , wherein the treatment to prevent CVD comprises administering a statin. 
     
     
         49 . The method of  claim 47 , wherein the one or more immune cell subpopulation is selected from CD4 T cells or subpopulations thereof, CD8 T cells or subpopulations thereof, intermediate monocytes or subpopulations thereof, B cells or subpopulations thereof, and Natural Killer cells or subpopulations thereof. 
     
     
         50 . The method of  claim 47 , wherein the sample is a blood, serum, or plasma sample, and the biomarkers are detected as a protein or a nucleic acid. 
     
     
         51 . The method of  claim 47 , wherein the immune cell subpopulation consists essentially of T cells and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 biomarkers selected from: SELPLG; IFITM3; MYC; FYB; JUNB; IL32; SELL; CCR7; GIMAP5; TCF7; FAS; BCL6; TNFSF10; STAT3; CD27; BIRC3; IKZF1; PTPRC; CYTIP; IFITM2; CD2; LCK; IL7R; FOXO1; TRAC; CCA; CD69; ICOS; SLC2A3; LEF1; RGS1; IL4R; CD52; HOPX; GZMA; KLRG1; KLRD1; TIGIT; STAT1; KLRC1; LGALS1; KLRC4; CD63; SAMD3; S100A10; KLRK1; IL23R; CCL5; APOBEC3G; CD160; TARP; CD3D; CD8A; KLRD1; ITGA4; CX3CR1; VNN2; GZMA; CH3L2; or GIMAP5, and optionally the biomarkers are selected in the order listed. 
     
     
         52 . The method of  claim 47 , wherein the immune cell subpopulation consists essentially of classical monocytes and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 40 biomarkers selected from: CCL4; CCL3; SLC2A3; SOD2; SELPLG; CD14; LAP3; FYB; LYN; CD80; TNFSF10; TNFSF8; CSF3; CSF3R; CD36; NAIP; SELL; CLEC4E; IL8R; PTPRC; VCAN; ADGRE1; MNDA; DUSP1; FYB; DUSP2; LYZ; JUNB; SCREP1; DOCK8; CD300A; LAP3; TNFSF13; SDCP; MDX1; FYN; STAT6; IL1B; NAMPT; STAT3; IL6; IER3; TLR2; CD83; FYB; S100A9; TNFSF13B; CLEC4E; or ICAM1, and optionally the biomarkers are selected in the order listed. 
     
     
         53 . The method of  claim 47 , wherein the immune cell subpopulation consists essentially of intermediate monocytes and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 biomarkers selected from: PRDM1; TNF; CCL4; LYZ; DUSP1; IL1B; DUSP2; CCL3; IER3; or ICAM1, and optionally the biomarkers are selected in the order listed. 
     
     
         54 . The method of  claim 47 , wherein the immune cell subpopulation consists essentially of B cells and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9 biomarkers selected from: CD74; IGHD; CD79A; LYN; CD83; R3HDM4; CD79B; CXCR5; or SLC2A3, and optionally the biomarkers are selected in the order listed. 
     
     
         55 . The method of  claim 47 , wherein the cardiovascular disease is selected from at least one of: coronary heart disease (CHD), heart failure (HF), coronary heart disease (CHD), cerebrovascular disease, peripheral arterial disease, atherosclerosis, or heart failure. 
     
     
         56 . A method of treating a subject that has or will develop a cardiovascular disease, the method comprising:
 (a) obtaining a sample from the subject;   (b) isolating immune cells from the sample;   (c) measuring a presence of one or more immune cell subpopulations from the isolated immune cells, and an expression level of one or more biomarkers in the one or more immune cell subpopulations;   (d) comparing the expression level of the one or more biomarkers in (c) with a statistical sample representative of the biomarkers in an equivalent immune cell subpopulation of a subject not having a cardiovascular disease; and   (e) ruling-out the subject as having cardiovascular disease from a diagnostic test for cardiovascular disease, a treatment of cardiovascular disease, or a combination thereof, based on the comparing step of (d), wherein if the expression level of the one or more biomarkers present in the subject sample is lower than a baseline level of the statistical sample, the subject is determined not to have or be likely to develop CVD; or   (f) administering a preventive treatment for the cardiovascular disease, a treatment for cardiovascular disease, or a combination thereof to the subject who is at risk of developing the cardiovascular disease based on the comparing step of (d), wherein if the expression level of the one or more biomarkers present in the subject sample is higher than a baseline level of the statistical sample, the subject is to have or be likely to develop CVD.   
     
     
         57 . The method of  claim 56 , wherein the one or more immune cell subpopulations is selected from CD4 T cells or subpopulations thereof, CD8 T cells or subpopulations thereof, intermediate monocytes or subpopulations thereof, B cells or subpopulations thereof, and Natural Killer cells or subpopulations thereof. 
     
     
         58 . The method of  claim 56 , wherein the sample is a blood, serum, or plasma sample, and the biomarkers are detected as a protein or a nucleic acid. 
     
     
         59 . The method of  claim 56 , wherein the immune cell subpopulation consists essentially of T cells and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, or 50 biomarkers selected from: SELPLG; IFITM3; MYC; FYB; JUNB; IL32; SELL; CCR7; GIMAP5; TCF7; FAS; BCL6; TNFSF10; STAT3; CD27; BIRC3; IKZF1; PTPRC; CYTIP; IFITM2; CD2; LCK; IL7R; FOXO1; TRAC; CCA; CD69; ICOS; SLC2A3; LEF1; RGS1; IL4R; CD52; HOPX; GZMA; KLRG1; KLRD1; TIGIT; STAT1; KLRC1; LGALS1; KLRC4; CD63; SAMD3; S100A10; KLRK1; IL23R; CCL5; APOBEC3G; CD160; TARP; CD3D; CD8A; KLRD1; ITGA4; CX3CR1; VNN2; GZMA; CH3L2; or GIMAP5, and optionally the biomarkers are selected in the order listed. 
     
     
         60 . The method of  claim 56 , wherein the immune cell subpopulation consists essentially of classical monocytes and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, or 40 biomarkers selected from: CCL4; CCL3; SLC2A3; SOD2; SELPLG; CD14; LAP3; FYB; LYN; CD80; TNFSF10; TNFSF8; CSF3; CSF3R; CD36; NAIP; SELL; CLEC4E; IL8R; PTPRC; VCAN; ADGRE1; MNDA; DUSP1; FYB; DUSP2; LYZ; JUNB; SCREP1; DOCK8; CD300A; LAP3; TNFSF13; SDCP; MDX1; FYN; STAT6; IL1B; NAMPT; STAT3; IL6; IER3; TLR2; CD83; FYB; S100A9; TNFSF13B; CLEC4E; or ICAM1, and optionally the biomarkers are selected in the order listed. 
     
     
         61 . The method of  claim 56 , wherein the immune cell subpopulation consists essentially of intermediate monocytes and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 biomarkers selected from: PRDM1; TNF; CCL4; LYZ; DUSP1; IL1B; DUSP2; CCL3; IER3; or ICAM1, and optionally the biomarkers are selected in the order listed. 
     
     
         62 . The method of  claim 56 , wherein the immune cell subpopulation consists essentially of B cells and the one or more biomarkers are selected from 1, 2, 3, 4, 5, 6, 7, 8, or 9 biomarkers selected from: CD74; IGHD; CD79A; LYN; CD83; R3HDM4; CD79B; CXCR5; or SLC2A3, and optionally the biomarkers are selected in the order listed. 
     
     
         63 . The method of  claim 56 , wherein the cardiovascular disease is selected from at least one of: coronary heart disease (CHD), heart failure (HF), coronary heart disease (CHD), cerebrovascular disease, peripheral arterial disease, atherosclerosis, or heart failure. 
     
     
         64 . The method of  claim 56 , wherein the method further comprises referring the subject to a specialist in cardiovascular disease. 
     
     
         65 . The method of  claim 38 ,  claim 47 , or  claim 56 , wherein step (b) is optionally omitted, and the immune cell subpopulations are measured directly from the sample.